The objectives of this study are to improve immunocytochemical methodology, to apply it to localize cell bodies of luteinizing hormone-releasing hormone (LHRH) neurons in the hypothalamus, and to characterize the catecholamine afferents to these cell bodies and their terminals in the median eminence (ME) and organum vasculosum of the lamina terminalis (OVLT). Antibodies to LHRH and the unlabeled antibody enzyme (peroxidase anti-peroxidase or PAP) technique of immunocytochemistry will be employed in a modified pre-embedding staining procedure which allows both light and electron microscopic study of the same immuno-positive structures. LHRH concentrations in cell bodies will be enhanced by using animals during the follicular phase, following ovariectomy, or after surgical deafferentation will also be used to eliminate certain monoaminergic inputs from areas of study and thus permit catecholamine neuron localization with antibodies to catecholamine synthesizing enzymes. LHRH antibodies and antibodies to tyrosine hydroxylase (TH), dopamine-B-hydroxylase (DBH), or phenylethanolamine-N-methyl transferase (PNMT) will be used in combination on the same tissue section. Using separate substrates giving different colored reaction products for each antigen, the relationship between LHRH neurons and those containing catecholamines will be examined at the light and then at the electron microscopic level. Quantification of LHRH by radioimmunoassay and of dopamine (DA) and noradrenaline (NA) by the catechol-O-methyl transferase (COMT) assay will be performed on microdissected hypothalamic areas and on the ME an OVLT. These data will be correlated with the results of the immunocytochemical localization.